In the United States, current screening programs for cervical cancer using the Papanicolaou (Pap) test are aimed at early detection and treatment of precancerous high-grade squamous intraepithelial lesions (HSIL). Of the greater than 3.5 million women who have equivocal or mild cytologic abnormalities on Pap tests each year, nearly 0.5 million have underlying HSIL. These findings highlight the limitations of the Pap test alone for the detection of HSIL. Testing for oncogenic HPV DNA is not an effective triage test in equivocal cases of atypical squamous cells, cannot exclude HSIL (ASC-H) and low-grade SIL (LSIL) due to the high prevalence of oncogenic HPV (>80%) and, as a result, all women with ASC-H and LSIL are referred to colposcopy. The risk of overtreatment and unnecessary costs related to this management strategy underscore the need for additional ancillary tests to improve the detection of HSIL in Pap tests. Aberrant DNA promoter methylation of tumor suppressor genes (TSGs) is an epigenetic alteration that contributes to tumor progression. Methylation of TSGs, such as DAPK1 and IGSF4, has been detected in >50% of precancerous HSIL and cervical cancers. Identification of tumor-specific methylation profiles and the availability of sensitive and specific PCR-based detection methods underlie its potential use as a molecular biomarker. The overall goal of this application is to determine if aberrant DNA methylation of TSGs can serve as a molecular biomarker of HSIL in liquid-based Pap tests to improve early detection of HSIL. The specific aims of this study are to 1) Develop a quantitative methylation-specific PCR (Q-MSP) assay for TSGs that distinguishes HSIL from LSIL and negative liquid-based Pap tests and 2) Determine if equivocal and mildly abnormal liquid-based Pap tests with underlying HSIL are positive forTSG methylation by the Q-MSP assay. For aim 1, we will use real-time quantitative PCR combined with standard MSP methods and residual liquid- based Pap tests to develop a Q-MSP assay for TSGs, including DAPK1 and IGSF4. For aim 2, we will apply the optimized Q-MSP assay to ASC-H and LSIL residual liquid-based Pap tests that have biopsy-confirmed HSIL and use statistical methods to analyze the assay performance. Development of a molecular biomarker that aids in the detection of HSIL in Pap tests that present diagnostic and management challenges forms the basis for future clinical trials and could have a major impact on cervical cancer screening. [unreadable] [unreadable] [unreadable]